Helicobacter pylori (H. pylori) is believed to infect about half of the world’s population and is related to many gastric diseases, duodenal ulcer and a variety of non-gastrointestinal tract disorders. In this study, the inhibitory effects of refined deep seawater (RDSW) on H. pylori growth and motility were investigated. Deep seawater was collected offshore from Muroto, Kochi, Japan, desalinated, and refined to increase the mineral concentration. The in vitro assays with 16 H. pylori isolates by 20 types of RDSW with combinations of 5 Mg/Ca ratios and 4 degrees of hardness were performed. Bacterial growth and mobility were inhibited in a hardness-dependent manner. A difference in the Mg/Ca ratio influenced the inhibitory effects when the degree of hardness was unchanged. At a hardness of 1000, anti-H. pylori activities were observed in all strains for at least at one of the 5 Mg/Ca ratios. Evaluation of the anti-H. pylori effects was then conducted in vivo in Mongolian gerbils, to which RDSW at 5 different Mg/Ca ratios (hardness: 1000) was administered for 2 weeks. The number of H. pylori colonized in the stomach showed a significant decrease with 2 types of RDSW (C and D), compared with commercial mineral water. Furthermore, 1 L of each 5 types of RDSW was given daily for 10 days to healthy subjects infected with H. pylori. An urea breath test examined before and after the 10-day period showed reduction of Δ13C values in more of these subjects compared to controls who drank commercial mineral water. In vivo anti-H. pylori effects were observed in ≥90% of subjects drinking any of the 5 types of RDSW. These results raised the possibility that the application of refined deep seawater can be available for prevention of or as an adjuvant therapy for H. pylori infection.